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Open Access Full Text Article ORIGINAL RESEARCH Prognostic Significance of CDH1, FN1 and VIM for Early Recurrence in Patients with Colorectal Liver Metastasis After Liver Resection

This article was published in the following Dove Press journal: Cancer Management and Research

Aleksandar Bogdanovic 1,2 Purpose: There are limited data on expression of epithelial–mesenchymal transition (EMT) Jovana Despotovic3 markers in patients with colorectal liver metastases (CRLM). The study aim was to evaluate Danijel Galun1,2 the expression and prognostic significance of E-cadherin (CDH1), fibronectin (FN1) and Nemanja Bidzic1,2 vimentin (VIM) in patients with CRLM after curative-intent liver resection. Aleksandra Nikolic3 Patients and Methods: Thirty patients with CRLM managed by curative-intent liver resection were included in this prospective pilot study. Blood samples, colorectal liver Jovana Rosic 2 metastases and surrounding non-tumor liver tissue were collected. Expression of CDH1, Zoran Krivokapic1,2,4 FN1 and VIM was analyzed by quantitative real-time polymerase chain reaction. Expression 1HPB Unit, Clinic for Digestive Surgery, in CRLM and non-tumor liver tissue was compared, while expression in serum was corre­ Clinical Center of Serbia, Belgrade, 11 lated with CRLM expression. One-year recurrence-free survival was compared between 000, Serbia; 2School of Medicine, University of Belgrade, Belgrade 11 000, patients with low and high CDH1, FN1 and VIM expression. 3 Serbia; Laboratory for Molecular Results: The expression of CDH1 was similar in CRLM and non-tumor liver tissues, while Biology, Institute of Molecular Genetics and Genetic Engineering, University of FN1 and VIM expression was significantly lower in metastatic tissue (P=0.003 and Belgrade, Belgrade, 11 000, Serbia; pP<0.001, respectively). Serum expression of CDH1 and VIM was detected in 66.7% and 4 Serbian Academy of Sciences and Arts, 93.3% of patients, respectively, while FN1 was not detected in any of the patients. The Belgrade 11 000, Serbia correlation of CDH1 and VIM expression between CRLM and serum was not statistically significant. Decreased CDH1 expression in CRLM and decreased VIM expression in serum were associated with early recurrence after surgical treatment of CRLM. Conclusion: Lower expression of CDH1 in CRLM and lower serum expression of VIM were found to be associated with early recurrence after liver resection for CRLM. Keywords: epithelial–mesenchymal transition, CDH1, VIM, FN1, colorectal liver metastasis, colorectal cancer

Introduction Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide.1 In 2017, 1.8 million new cases of CRC were diagnosed and 896,000 deaths were registered.2 CRC is characterized by growing incidence and increasing mortality rates in developing countries, mostly in Eastern Europe, Asia and South America.3 4 Correspondence: Aleksandar Bogdanovic Liver is the most common metastatic site of colorectal cancer. Approximately Clinical Center of Serbia – Clinic for 50% of CRC patients develop liver metastases during the course of the disease.5 Digestive Surgery, Koste Todorovica 6, 6 Belgrade 11 000, Serbia Currently, liver resection is the only potentially curative treatment option. Tel +381 64 2461104 Although long-term survival has been substantially improved, more than half of Email aleksandarbogdanovic81@yahoo. 7 com patients will develop recurrent disease two years after liver resection. The majority submit your manuscript | www.dovepress.com Cancer Management and Research 2021:13 163–171 163 DovePress © 2021 Bogdanovic et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/ http://doi.org/10.2147/CMAR.S287974 terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Bogdanovic et al Dovepress of patients are managed by perioperative chemotherapy, investigated. The prognostic value of EMT markers in associated with different side-effects. prediction of rapid disease progression after curative- Long-term prognosis of patients with colorectal cancer intent treatment is still not elucidated. liver metastases (CRLM) is uncertain, while prediction of This study was designed as a pilot study on a limited chemotherapy response is equally difficult. Several predic­ number of patients with the aim to analyze gene expres­ tive scoring systems were developed to stratify prognosis sion status of CDH1, FN1 and VIM in serum, CRLM and and subsequently allocate the optimal treatment non-tumor liver tissue, and to evaluate their eventual prog­ modality.8,9 These scoring systems are clinically applic­ nostic significance for early recurrence after curative- able in terms of survival but have limited predictive value intent liver resection for CRLM. in terms of patient stratification for clinical management.10 Moreover, patients with similar clinical stages may Patients and Methods achieve different survival. This reflects the high degree of biological heterogeneity of CRLM, responsible for dif­ Subjects Between December 2016 and February 2019, thirty ferences in prognosis and responsiveness to therapy. In the patients with CLRM were recruited for the prospective era of personalized medicine based on genomic analysis pilot study. All subjects were managed by curative-intent and targeted therapies, there is an urgent need for new liver resection at University Clinic for Digestive Surgery, clinically useful biomarkers. Their use should aid patient Clinical Center of Serbia, Belgrade. The inclusion criteria stratification and selection of tailored cancer treatment were age 18–90 and potentially curative hepatectomy for strategies to individual patients.11 CRLM, simultaneously or after radical resection of Epithelial–mesenchymal transition (EMT) has an a primary tumor. The exclusion criteria were previous important role in promoting tumor invasion and metastasis hepatectomy for CRLM and the presence of residual extra­ of cancers with epithelial origin.12 EMT is a process by hepatic disease. This study was conducted in accordance which epithelial cells lose their cell-cell adhesion and with the Declaration of Helsinki. The study was approved polarity, acquiring a mesenchymal phenotype with by the Ethics Committee of Clinical Center of Serbia. increased migratory capacity.13 In recent years, EMT has Informed consent was obtained from all individual parti­ emerged as well-recognized underlying molecular cipants included in the study. mechanism in a variety of cancers, including CRC.14–16 Preoperative tumor staging was determined by transab­ The process of EMT is associated with decreased expres­ dominal ultrasound, chest radiography, computed tomo­ sion of epithelial markers such as E-cadherin (CDH1), and graphy (CT) and/or magnetic resonance imaging (MRI). increased expression of mesenchymal markers, such as Demographic and clinicopathological features were fibronectin (FN1) and vimentin (VIM).17 E-cadherin is recorded: age, sex, primary cancer data (localization, a transmembrane protein and the core component of neoadjuvant/adjuvant chemo-radiotherapy, TNM and epithelial adherens junctions, essential for tissue develop­ Duke’s classification), number, diameter and lobar distri­ ment, differentiation, and maintenance.18 The loss of bution of liver metastases, carcinoembryonic antigen E-cadherin during the EMT process leads to destroyed (CEA) and carbohydrate antigen (CA 19–9), perioperative cell-cell adhesion, increased cell motility and advanced chemotherapy, and tumor grading and tumor residual stages of cancer.19 Fibronectin is an extracellular matrix status. glycoprotein, having an important role in cell adhesion and migration and affecting cell proliferation.20 Vimentin, as a type III intermediate filament, has a role in maintaining Samples cytoskeleton organization and focal adhesion stability.21 Tumor tissue and surrounding non-tumor liver tissue sam­ Several studies have investigated expression of indivi­ ples were obtained from resected liver dual markers of EMT in primary CRC, and confirmed their specimens immediately after surgery. Tissue samples potential prognostic role.22,23 However, there are limited were immersed into RNAlater® RNA Stabilization data about EMT genes expression in patients with Solution (ThermoFisher Scientific, Vilnius, Lithuania) CRLM.24,25 Moreover, relation and correlation between and stored at −80°C. Corresponding blood samples were EMT genes expression in serum, metastatic CRC tissue obtained one week prior to surgery. Blood samples were or non-tumor liver tissue have been insufficiently allowed to coagulate for 15 min, and then centrifuged at

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3500 rpm for 10 min. Obtained sera were stored at −80°C Quantitative Real-Time Polymerase Chain until extraction of RNA. Reaction (qRT-PCR) qRT-PCR was performed using HOT FIREPol® EvaGreen® RNA Extraction qPCR Mix Plus (ROX) (Solis BioDyne, Tartu, Estonia). Collected tumor tissue and non-tumor liver tissue was cut into Primers used for EMT genes were: CDH1 forward 5ʹ- smaller pieces and homogenized prior to addition of 1 mL of TGCCCAGAAAATGAAAAAGG-3ʹ, CDH1 reverse 5ʹ-GT TRIzol Reagent (Thermo Fisher Scientific, Vilnius, GTATGTGGCAATGCGTTC-3ʹ, FN1 forward 5ʹ-CAGTGG Lithuania). Total RNA from tissue was extracted according GAGACCTCGAGAAG-3ʹ, FN1 reverse 5ʹ-TCCCTCGGA to the manufacturer’s protocol, while the RNA from serum ACATCAGAAAC-3ʹ, VIM forward 5ʹ-GAGAACTTTGCC was extracted using the following protocol. 1 mL of TRIzol GTTGAAGC-3ʹ, VIM reverse 5ʹ-GCTTCCTGTAGGTGGC Reagent was added to 400 μL of serum together with 1 μL of AATC-3ʹ.26 The expression of glyceraldehyde-3-phosphate 1 μg/μL nuclease-free glycogen. The mixture was vortexed dehydrogenase (GAPDH) housekeeping gene was used as for 20 s to dissipate the yellow globules and incubated at an endogenous control for normalization of CDH1, FN1 and room temperature for 10 min. Then, 200 μL of chloroform VIM relative gene expression. GAPDH primers were was added. The tube was shaken vigorously by hand for 20 GAPDH forward 5ʹ-GTGAAGGTCGGAGTCAACG-3ʹ simmediately after chloroform addition, incubated for 15 min and GAPDH reverse 5ʹ-TGAGGTCAATGAAGGGGTC-3ʹ. at room temperature and centrifuged at 12,000 x g for 15 min qRT-PCR was carried out in a 7500 Real-Time PCR System at 4°C. Approximately 800 μL of the upper aqueous phase machine (Applied Biosystems, Foster City, California, was transferred to a fresh tube, followed by the addition of USA). PCR conditions were as follows: 50°C for 2 min, 1.2 mL of isopropanol. Mixture was incubated at room tem­ 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and perature for 10 min and centrifuged at 12,000 x g for 8 min at 60°C for 60 s. The qRT-PCR reaction for each sample was 4°C. The supernatant was carefully aspirated and 1 mL of done in triplicate. Data were extracted and analyzed using 75% ethanol was added. The tube was centrifuged at 7500 x g 7500 System Software. Data are presented as average ΔCt for 5 min at room temperature. The supernatant was removed values. and the pellet was allowed to dry for 5 min. The RNA pellet was resuspended with 20 μL of nuclease-free water and Follow Up stored at −80°C. The concentration and purity of total RNA Patients were followed up during the first postoperative year from tissue and serum were determined by measuring the after liver resection. Tumor markers CEA and CA 19–9 and absorbance at 260 nm and 280 nm. transabdominal ultrasonography were performed every three months. Computed tomography and/or magnetic resonance Gene Expression Analysis were performed six months and one year after liver resection. Extracted total RNA was treated with Ambion™ DNase Colonoscopy/rectoscopy and positron emission tomography I (RNase free) (ThermoFisher Scientific, Vilnius, were considered when local or distant recurrence was sus­ Lithuania) in the presence of the 10 x DNase Buffer and pected. Early recurrence was defined by the presence of incubated at 37°C for 30 min. DNase I was inactivated by either local recurrence (at the site of primary cancer resec­ 2 min incubation with DNase Inactivation Reagent from tion), appearance of new liver metastases or extrahepatic the DNA-free™ DNA Removal Kit (ThermoFisher disease during the first year after surgery. According to the Scientific, Vilnius, Lithuania). The mixture was centri­ status of recurrent disease, patients were further stratifiedinto fuged at 10,000 x g for 1.5 min and the supernatant recurrence and no recurrence groups. containing DNA-free RNA was transferred to a new tube. The total RNA was reverse-transcribed using the High Statistical Analysis Capacity cDNA Reverse Transcription Kit (Applied Statistical analysis was carried out using SPSS version Biosystems, Vilnius, Lithuania) according to the manufac­ 18.0 (SPSS Inc., Chicago, IL, USA). P value ≤0.05 was turer’s instructions. The reverse-transcription mixture was considered statistically significant. Continuous variables incubated for 10 min at 25°C, 120 min at 37°C and 5 min were expressed as median (range). Categorical variables at 85°C. Complementary DNA (cDNA) was stored at were expressed as absolute numbers (percentages). −20°C until further use. Normal distribution of continuous data was tested using

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Kolmogorov–Smirnov normality test. If data were nor­ Table 1 Patients’ Characteristics mally distributed, Student’s t-test was applied, and Study Patients (n=30) Pearson bivariate correlation coefficient was determined. Age (years), median (range) 67.5 (24–82) If not, Mann–Whitney U-test for two independent samples or Wilcoxon signed ranks test for two related samples Sex, n (%) were used. Non-parametric Spearman’s rank correlation Male 16 (53.3) Female 14 (46.7) was determined for non-normal distributions. Receiver operating characteristic (ROC) curves were Primary cancer data, n (%) created to determine optimal, sample-based cut-off values Site of CDH1, FN1 and VIM expression in CRLM and serum Colon 13 (43.3) by Youden index (smaller test results indicate more posi­ Rectum 17 (56.7) tive test for CRLM CDH1 and VIM expression and serum T stage VIM expression). Patients were stratified according to cut- T1-2 1 (3.3) off values into the appropriate low- and high- CDH1, FN1 T3-4 29 (96.7) and VIM groups. One-year recurrence-free survival of low- N stage and high- CDH1, FN1 and VIM groups was estimated by N0 8 (26.7) the Kaplan-Meier method and compared using Log rank N1-2 22 (73.3) test. Univariate analysis was performed to analyze prog­ Duke’s classification nostic factors affecting one year recurrence. Univariate A-B 3 (10) analysis was conducted using Fisher’s exact test for cate­ C-D 27 (90) gorical variables or Mann–Whitney U-test for continuous Neoadjuvant chemoradiotherapy 5 (16.7) variables. Multivariate analysis was not done since only Adjuvant chemotherapy 5 (16.7) univariate analysis found significant parameters. Colorectal liver metastases, n (%) Results Number The expression of CDH1, FN1 and VIM was analyzed in ≤3 19 (63.3) >3 11 (36.7) CRLM, non-tumor liver tissue and in serum from patients with hepatic metastases of CRC. Demographic and clin­ Lobar distribution icopathological data of the included subjects are summar­ Unilobar 15 (50) Bilobar 15 (50) ized in Table 1. Maximum diameter (mm) CDH1 ≤50 23 (76.7) CDH1 expression in CRLM was −2.28 (range, −7.39 to >50 7 (23.3) −0.81), in non-tumor liver tissue −2.64 (range, −4.48 to Time of diagnosis −1.33) and in serum −4.15 (range, −10.48 to −0.56). Synchronous 22 (73.3) Metachronous 8 (26.7) FN1 Preoperative chemotherapy 15 (50) FN1 expression in CRLM was −0.79 (range, −6.06–2.03) Histologic grade and in non-tumor liver tissue 2.75 (range, −6.73–4.75). Well differentiated (G1) 15 (50) Moderately differentiated (G2) 15 (50) VIM CEA (ng/mL), median (range) 27.5 (2–704) VIM expression in CRLM, non-tumor liver tissue and CA 19–9 (IU/mL), median (range) 37.5 (2–12,999) serum was −1.52 (range, −5.28–2.59), −0.8 (range, −2.­ Abbreviations: CEA, carcinoembryonic antigen; CA 19–9, carbohydrate antigen. 24–1.61) and −0.97 (range, −2.64–1.99), respectively. CDH1 expression in CRLM was similar to non-tumor C). Serum expression of CDH1 was detected in 66.7% of liver tissue (Figure 1A), while expression of FN1 and VIM patients, while VIM expression was detected in 93.3% of in CRLM was significantly lower than in non-tumor liver patients. Serum expression of FN1 was not detected in any tissue (P=0.003 and P<0.001, respectively) (Figure 1B and of the patients.

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Figure 1 Expression of FN1 and VIM is decreased in colorectal liver metastasis when compared with non-tumor liver tissue, while expression of CDH1 is similar. Expression of CDH1 (A), FN1 (B) and VIM (C) in CRLM and surrounding non-tumor liver tissue measured by qRT-PCR. Data are presented as average ΔCt values (median with range). **P <0.01; ***P <0.001.

Figure 2 Expression of CDH1 and VIM in CRLM does not correlate with their expression in serum. Scatterplot represents correlation of CDH1 (A) and VIM (B) expression in CRLM and serum, while the straight line represents linear regression model. r in (A), Spearman’s coefficient; r in (B), Pearson’s coefficient.

As shown in Figure 2, there was no significant correla­ off value for serum expression of CDH1 and VIM was −3.16 tion between expression of CDH1 (Spearman’s rho= (sensitivity 44.4%, specificity 81.8%) and 0.26 (sensitivity −0.255, P=0.322) or VIM (Pearson’s r=0.181, P=0.357) 92.3%, specificity 40%), respectively. According to esti­ in serum and CRLM (Figure 2A and B). mated cut-off values, patients were subsequently divided into corresponding low- and high- groups based on CRLM ROC Curve Analysis and Cut-off Values expression: 6 (24%) in low-CDH1 and 19 (76%) in high- Estimation CDH1, 14 (58.3%) in low-FN1 and 10 (41.7%) in high-FN1, An optimal cut-off value for CRLM expression of CDH1, and 10 (33.3%) in low-VIM and 19 (63.3%) in high-VIM; and FN1 and VIM was −3.59 (sensitivity 54.5%, specificity also according to serum expression: 14 (70%) in low-CDH1 99.9%), −0.54 (sensitivity 60%, specificity 71.4%) and and 6 (30%) in high-CDH1 and 21 (75%) in low-VIM and 7 −1.98 (sensitivity 50%, specificity60%), respectively. A cut- (25%) in high-VIM.

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Prognostic Factors of Early Recurrence selected based on previous reports that demonstrated clin­ Median one-year recurrence-free survival was 12 (range, ical relevance of CDH1, FN1 and VIM in CRC 27–29 4–12) months. Patients in high-CDH1 CRLM group patients. The major findings of this study are: experienced longer one-year recurrence-free survival than decreased expression of FN1 and VIM in CRLM in com­ patients in low-CDH1 CRLM group (P=0.001), while one- parison to non-tumor liver tissue, and association of year recurrence-free survival for FN1 and VIM was similar decreased CDH1 expression in CRLM and decreased in both groups (P=0.160 and P=0.108, respectively). VIM expression in serum with early recurrence after sur­ Based on serum analysis, high-VIM group showed longer gical treatment of CRLM. recurrence-free survival than low-VIM group (P=0.041), The majority of published studies focused on identify­ while recurrence-free survival was similar between high- ing potential protein biomarkers using immunohistochem­ istry analysis of tissue samples or using enzyme-linked and low-CDH1 groups (P=0.265). Kaplan–Meier curves immunosorbent assay to measure their plasma are shown in detail in Figure 3. concentrations.25,30 Recent attention was given to circulat­ With univariate analysis only CDH1 expression in CRLM ing cell-free nucleic acids (cfNAs), such as DNA, mRNA was found as a prognostic factor for one-year recurrence or microRNA (miRNA), and cellular nucleic acids prediction (P=0.041). Age more than 65, sex, neoadjuvant (cNAs). Currently, the most commonly used methods for chemotherapy, synchronous presentation, bilobar distribution, cfRNAs and cNAs detection are qRT-PCR, microarray and multiple metastases, number of metastases ≥3, tumor size deep sequencing.31 cfNAs originate from apoptotic and ≥50 mm, histologic grade G2, residual status R1, FN1 and necrotic cancer cells digested by macrophages and then VIM expression level in CRLM and CDH1 and VIM expres­ released into the circulation.32 cfNAs from the peripheral sion level in serum had no predictive ability for early recur­ blood are easily accessible and may serve as a minimal rence (Table 2). Therefore multivariate analysis was not done. invasive diagnostic tool – “liquid biopsy”, to replace sur­ gical soft tissue biopsy. Furthermore, cfNAs can be used Discussion as prognostic markers, during the follow-up in already The presented pilot study analyzed expression of three treated patients or to evaluate therapeutic response. genes involved in EMT using different tissue samples Higher concentrations of cfNAs have been registered and serum of patients with CRLM. A panel of genes was in cancer patients, although increased concentrations can

Figure 3 Decreased CDH1 expression in CRLM and decreased VIM expression in serum was associated with one-year recurrence following liver resection. Kaplan–Meier curves of one-year recurrence-free survival for low- and high- CDH1, FN1 and VIM groups based on CRLM and serum expression. CRLM, colorectal liver metastases.

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Table 2 Univariate Analysis of Potential Prognostic Factors Related to Early Recurrence After Liver Resection for Colorectal Liver Metastases

Recurrence n=14 (46.7%) No Recurrence n=16 (53.3%) P value

Age (˃65 years) 9 (64.3) 9 (56.3) 0.722 Sex (female) 7 (50) 7 (43.8) 1 Neoadjuvant chemotherapy 6 (42.9) 9 (56.3) 0.715 Synchronous presentation 12 (85.7) 10 (62.5) 0.226 Bilobar distribution 7 (50) 8 (50) 1 Multiple metastases 9 (64.3) 9 (56.3) 0.722 Number of metastases (≥3) 4 (28.6) 7 (43.8) 0.466 Tumor size (≥50mm) 3 (21.4) 4 (25) 1 Histologic grade (G2) 9 (64.3) 6 (37.5) 0.272 Residual status (R1) 9 (64.3) 11 (68.8) 1 CDH1 level in CRLM, median (range) −3.81 (−7.39- −0.94) −2.24 (−3.38- −0.81) 0.041 CDH1 level in serum, median (range) −3.50 (−10.48- −1.82) −4.30 (−10.39- −0.56) 0.973 VIM level in CRLM, median (range) −1.84 (−5.28–2.59) −1.22 (−3.42–0.28) 0.467 VIM level in serum, median (range) −1.09 (−2.64- −0.14) −0.71 (−2.48–2) 0.345 FN1 level in CRLM, median (range) −0.26 (−3.25–2.03) −0.95 (−6.05–1.79) 0.191

Abbreviation: CRLM, colorectal liver metastases. be found in other conditions, like inflammatory diseases or expression level tended to increase in CRLM, statistical benign tumors.33 To date, miRNA are extensively investi­ difference in comparison to non-tumor liver tissue expres­ gated in CRC patients and their prognostic role was con­ sion was not observed in our study. These findings are firmed in several studies.34,35 Unlike miRNAs, circulating consistent with previously described mesenchymal–epithe­ mRNAs are poorly studied as disease biomarkers. lial and epithelial–mesenchymal cellular plasticity of liver Although various molecules are involved in the process metastases.42 The other possible explanation for this find­ of EMT (i.e. CDH1, CDH2, TCF-8, Claudin-1, Zo-1, β- ing is a small sample size. catenin, Snail, etc.), the presented study is among the first We were unable to detect serum FN1 mRNA in any of that analyzed cfmRNAs for CDH1, VIM and FN1 as our patients. Inability to detect FN1 mRNA in serum is not metastatic CRC prognostic biomarkers. related to technical constraints, since GAPDH was success­ Decreased levels of tumor tissue mRNA for VIM and fully detected in all patients in tissue samples and in serum, FN1 were observed in CRLM compared with non-tumor confirming the sample quality. Also, FN1 mRNA was suc­ liver tissue. Niknami et al. showed up-regulated VIM and cessfully detected in our tissue samples which eliminates FN1 expression in stromal cells, while VIM was down- the possibility of poor primer selection. This could mean regulated in colonic epithelial cells.36 Niknami et al. that FN1 mRNA is either so low that it cannot be detected further demonstrated an association between decreased by qRT-PCR, or is completely absent in serum of mCRC mRNA expression of VIM and larger tumor size, and patients, probably because of low stability of FN1 mRNA. increased FN1 levels and higher tumor stages. Chaffer Nevertheless, fibronectin exists in serum of CRC patients et al. proposed that mesenchymal–epithelial transition and it is a useful marker of disease advancement.43 (MET) occurred during the invasion of targeted Serum CDH1 and VIM were not found to reflect their organs by bladder cancer, and that cells start acquiring expression in CRLM, although this result was expected epithelial phenotype, showing a decreased vimentin considering the shedding of tumor cells into the circula­ expression.37 Other authors have also reported that MET tion. Despite no correlation being confirmed by the current seems to occur after invasion of targeted organs and for­ analysis, the association between serum and malignant mation of distant metastases.38,39 Truant et al. and Ionescu tissue expression should be further investigated, since et al. showed similar E-cadherin expression in colon can­ cfmRNAs for CDH1 and VIM might have important cer and adjacent normal mucosa.40,41 However, Truant roles in CRLM patients. et al. reported E-cadherin expression was higher in liver The presented study confirmed the prognostic signifi­ metastases than in non-tumor liver tissue. Although CDH1 cance of CDH1 expression in CRLM tissue for the

Cancer Management and Research 2021:13 submit your manuscript | www.dovepress.com 169 DovePress Bogdanovic et al Dovepress prediction of early recurrence. The majority of studies Disclosure have investigated the prognostic impact of EMT markers The authors report no conflicts of interest in this work. in primary cancers after curative-intent surgery. Several authors reported a relationship between hepatic metastatic tissue gene expression and long-term prognosis after liver References resection.25,44,45 Nanashima et al. analyzed expression of 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence E-cadherin in hepatic metastases of colorectal carcinoma and mortality worldwide for 36 cancers in 185 countries. CA Cancer by immunohistochemistry, and found that patients with J Clin. 2018;68(6):394–424. doi:10.3322/caac.21492 preserved expression of E-cadherin had better prognosis 2. Fitzmaurice C, Abate D; Global Burden of Disease Cancer C. Global, Regional, and National Cancer Incidence, Mortality, Years of Life compared with those with negative expression, although Lost, Years Lived With Disability, and Disability-Adjusted the difference was not significant.44 The same author Life-Years for 29 Cancer Groups, 1990 to 2017: A Systematic Analysis for the Global Burden of Disease Study. JAMA Oncol. reported no association between E-cadherin levels and 2019. doi:10.1001/jamaoncol.2019.2996 recurrence-free survival period in a larger sample size 3. Arnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, analysis.45 Also, immunohistochemically analyzed, Bray F. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66(4):683–691. doi:10.1136/gutjnl-2015-310912 E-cadherin levels in metastatic liver tissue were not 4. Park JH, Kim JH. Pathologic differential diagnosis of metastatic found to be associated with survival.25 We estimated carcinoma in the liver. Clin Mol Hepatol. 2019;25(1):12–20. doi:10.3350/cmh.2018.0067 expression of mRNAs for CDH1, VIM and FN1 by qRT- 5. Adam R, Hoti E, Folprecht G, Benson AB. Accomplishments in 2008 PCR and observed an association between CRLM CDH1 in the management of curable metastatic colorectal cancer. level and early recurrence. This study also reports pro­ Gastrointest Cancer Res. 2009;3(5 Supplement 2):S15–22. 6. Chow FC-L, Chok KS-H. Colorectal liver metastases: an update on longed recurrence-free survival in patients with high-VIM multidisciplinary approach. World J Hepatol. 2019;11(2):150–172. serum expression, but no difference between the study doi:10.4254/wjh.v11.i2.150 7. de Jong MC, Pulitano C, Ribero D, et al. Rates and patterns of groups was observed. recurrence following curative intent surgery for colorectal liver The small sample size of this study is a major study limit metastasis: an international multi-institutional analysis of 1669 and should be overcome by conducting larger and multicenter patients. Ann Surg. 2009;250(3):440–448. doi:10.1097/SLA.0b013e3 181b4539b studies. However, the pilot study was designed as the firststep 8. Sasaki K, Morioka D, Conci S, et al. The Tumor Burden Score: aimed to assess prognostic significance of CDH1, VIM and A New “Metro-ticket” Prognostic Tool For Colorectal Liver Metastases Based on Tumor Size and Number of Tumors. Ann FN1 for early recurrence after curative-intent liver resection. Surg. 2018;267(1):132–141. doi:10.1097/SLA.0000000000002064 Furthermore, long-term survival was not included in the cur­ 9. Fong Y, Fortner J, Sun RL, Brennan MF, Blumgart LH. Clinical score rent analysis, since this study is designed as a pilot project. for predicting recurrence after hepatic resection for metastatic color­ ectal cancer: analysis of 1001 consecutive cases. Ann Surg. 1999;230 Thorough evaluation of EMT gene expression as biomarkers (3):309–318. doi:10.1097/00000658-199909000-00004 will also require long-term follow up of multiple clinical and 10. Gomez D, Cameron IC. Prognostic scores for colorectal liver metas­ histopathological parameters. tasis: clinically important or an academic exercise? HPB (Oxford). 2010;12(4):227–238. doi:10.1111/j.1477-2574.2010.00158.x In summary, the presented pilot study confirmed prog­ 11. Aziz MA, Yousef Z, Saleh AM, Mohammad S, Al Knawy B. nostic significance of CDH1 expression in CRLM tissue Towards personalized medicine of colorectal cancer. Crit Rev Oncol Hematol. 2017;118:70–78. doi:10.1016/j.critrevonc.2017.08. for prediction of early recurrence. 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